Treatment to reduce edema

ABSTRACT

Administration of a corticotropin-releasing factor (or a salt or analog thereof) decreases the leakage of blood components into tissues produced by various adverse medical conditions. Thus, treatments with corticotropin-releasing factor are useful in systemic inflammatory conditions.

This is a division of Ser. No. 08/229,911, filed Apr. 19, 1994 now U.S.Pat. No. 5,488,033, issued, Jan. 30, 1996, which is acontinuation-in-part of Ser. No. 07/876,487, filed Apr. 30, 1992, nowU.S. Pat. No. 5,306,710, issued Apr. 26, 1994, which is a divisional ofSer. No. 07/386,885, filed Jul. 28, 1989, now U.S. Pat. No. 5,137,871,issued Aug. 11, 1992.

FIELD OF THE INVENTION

This invention generally relates to a method of reducing edema inconnection with brain and musculature injuries, and more particularly tothe use of corticotropin-releasing factor or its analogs in reducingedema of the brain and musculature following injury to or disease ofthese vascular beds and in treating severe inflammatory conditions, suchas systemic inflammatory response syndrome, which results from e.g.,sepsis, hemorrhagic shock, tissue injury.

This invention was made with Government support under Grant No. DA-00091awarded by the National Institutes of Health. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Inflammation is signaled by redness, swelling, heat, and pain as areaction of the body against injury or assault. A variety of chemicalshave been implicated as chemical mediators of the inflammatory reaction,including histamine, serotonin, kinins, prostaglandins,platelet-activating factors, leukotrienes, and, from nerve endings,substance P. Mediators of the acute inflammatory reaction seem to playroles in one or more of increasing vascular permeability, attractingleukocytes, producing pain, local edema, and necrosis.

A variety of physiologic responses occur from the biological events thatconstitute the inflammatory processes. For example, Pinckard et al. atChapter 10 describe platelet-activating factors ("PAF") in the textInflammation: Basic Principles and Clinical Correlates (Gallin et al.Ed., 1988). This family of structurally related compounds appear topromote a variety of physiologic actions that are directly or indirectlyrelated to inflammatory reactions. The authors note that PAF has beenimplicated in the pathogenesis of human disease conditions such asendotoxin shock and organ transplantation rejection.

There are steroid and non-steroid, anti-inflammatory drugs known to theart. U.S. Pat. No. 4,579,844, inventors Rovee et al., issued Apr. 1,1986, discloses topically treating an inflammatory condition of the skinby use of the prostaglandin synthetase inhibitor concurrently with acorticosteroid. U.S. Pat. No. 4,404,198, inventor Kelley, issued Sep.13, 1983, discloses the topical application of a composition includingphenyl salicylate to treat inflammation. U.S. Pat. No. 3,980,778,inventors Ayer et al., issued Sep. 14, 1976, discloses asteroid for usein the topical, oral or parenteral treatment of skin and mucous membraneinflammations. Ibuprofen (a known anti-inflammatory agent) has beentested in connection with UV-B-induced inflammation, but was found tohave limited usefulness in treating sunburn reaction and is onlysomewhat more effective than placebo for the relief of symptomsassociated with UV-B-induced inflammation after high dose UV-Bphototherapy for psoriasis. Stern et al., Arch. Derm., 121, pp. 508-512(1985).

U.S. Pat. No. 4,801,612, inventor Wei, issued Jan. 31, 1989, disclosesthe use of inhibiting an inflammatory response in the skin or mucosalmembranes of a patient by administering corticotropin-releasing factor,or its analogs.

However, the microcirculation for mammals has its own selectivepharmacology for each particular vascular bed. This means that ananti-inflammatory agent useful in one vascular bed, such as the skin andmucosal membranes, cannot predictably be useful with other vascularbeds, such as the brain or musculature. For example, histamine,bradykinin, serotonin, or arachidonic acid failed to increasepermeability in blood vessels of the pia mater (the innermostvascularized covering of the brain), although these substances arepotent edema producing agents in the skin and mucosa. Another example ofselective pharmacology is epinephrine, since this endogenous substanceconstricts blood vessels in the skin but dilates blood vessels inskeletal muscle. Thus, the permeability characteristics of the bloodvessels (particularly the post-capillary venules) in a vascular bed suchas the brain are not equivalent to those in the skin and mucosa.

Corticotropin-releasing factor (hereinafter "CRF") is a 41 amino acidneuropeptide that is present in brain and the peripheral nerve endings,and stimulates ACTH release from pituitary cells. U.S. Pat. No.4,489,163, inventors Rivier et al., issued Dec. 18, 1984, discloses ratCRF and its analogs. Human CRF has the same sequence as rat CRF.

There are a number of analogs of CRF known to the art. U.S. Pat. No.4,415,558, inventors Vale, Jr. et al., issued Nov. 15, 1983, disclosesthe synthesis of sheep CRF, analogs, and isolation of the oCRF fromovine hypothalamic extracts. The synthetic OCRF was found to lower bloodpressure.

A generally similar peptide, sauvagine, was described in RegulatoryPeptides 2, 1-13 (1981). Sauvagine is a 40 amino acid peptide and hasbeen reported to have biological activity in lowering blood pressure inmammals and stimulating the secretion of ACTH and β-endorphin.

U.S. Pat. No. 4,528,189, inventors Lederis et al., issued Jul. 9, 1985,and U.S. Pat. No. 4,533,654, inventors Lederis et al., issued Aug. 6,1985, disclose peptides similar to the rat and sheep CRF and analogsthereof, and found this white sucker and carp urotensin respectively tostimulate ACTH and to lower blood pressure.

The other CRF-related peptide, white sucker urotensin, has an amino acidsequence the same as the carp urotensin, except the amino acid at the 24position is isoleucine and the amino acid at the 27 position is glutamicacid.

Ling et al., BBRC, 122, pp. 1218-1224 (1984), disclose the structure ofgoat CRF, which is the same as that for sheep CRF. Esch et al., BBRC,122, pp. 899-905 (1984), disclose the structure of bovine CRF whichdiffers from sheep and goat CRF only by one amino acid residue (number33 which is Asparagine rather than the number 33 Serine of goat andsheep CRF). Porcine CRF has been isolated and characterized by Patthy etal., Proc. Natl. Acad. Sci., 82, pp. 8762-8766 (1985). Porcine CRFshares a common amino acid sequence (residues 1-39) with rat/human CRFand differs from these only in position 40 and 41. Residue 40 can beeither asparagine or isoleucine and residue 41 is phenylalanine-amide.

SUMMARY OF THE INVENTION

In one aspect of the present invention, a method of treating a patientfor a systemic inflammatory response syndrome comprises administering tothe patient CRF (or a salt or analog thereof). Patients are treated fora systemic inflammatory response syndrome resulting from conditions suchas sepsis.

Administration in accordance with the invention also reduces thepermeability of brain and central nervous system blood vessels and is oftherapeutic value in the treatment of brain and central nervous systeminjuries. Thus, for example, the serious medical emergency posed bybrain edema, where the increased amounts of water compress and distorttissue architecture and impede delivery of oxygen to brain cells, can besubstantially avoided or alleviated. Administrations in accordance withthe invention also provide clinical benefits when used to limit orminimize leakage of blood constituents into tissue during surgery.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows two rat brains one hour after injury. The cortex stainedwith a blue dye (shown by shading, delineates the area of increasedvascular permeability produced by cold injury. On the left is the brainof a rat treated with saline. On the right is the brain of a rat treatedin accordance with the invention;

FIG. 2 shows two rat muscle tissue sections taken 1/2 hour after muscleinjury (a 4 cm mid-line incision, or celiotomy). The tissue stained witha blue dye (shown by shading delineates the area of increased vascularpermeability due to the surgical injury. On the left is the tissue of arat treated with saline. On the right is the tissue of a rat treated inaccordance with the invention; and

FIG. 3 graphically illustrates the activity of corticotropin-releasingfactor in the neutropenic rat model.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Systematic inflammatory response syndrome is the designation recentlyestablished by a group of researchers to describe related conditionsresulting from, for example, sepsis, pancreatitis, multiple trauma suchas injury to the brain, and tissue injury, such as laceration of themusculature, brain surgery, hemorrhagic shock, and immune-mediated organinjuries.

When an injury to the brain occurs, such as brain ischemia, orinfarction, vasogenic edema occurs and the increased amounts of watercompress and distort brain tissue architecture and impede the deliveryof oxygen to brain cells. The patient can lose consciousness and stopbreathing. I have discovered that CRF, its analogs, and related peptides(e.g., sauvagine and urotensin I) are effective in reducing theleakiness in the blood vessels of the brain (technically quantified as achange in vascular permeability) after injury. This discovery wassurprising because the blood vessels of the brain, in contrast to thevessels found in the skin and mucosa, appear to have "tighter"junctions, and normally do not respond to the inflammatory mediatorsthat promote leakage of blood vessels in the skin.

By "CRF" is meant herein mammalian corticotropin-releasing factor,including that isolatable from rat, human, beef, goat, pig, or sheep.Analogs of CRF include sauvagine, carp urotensin and sucker urotensin(all of which have been isolated from lower vertebrates), and thosesynthetic peptide structures analogous to CRF and disclosed in U.S. Pat.Nos. 4,415,558, 4,489,163, 4,553,654, and 4,528,189, incorporated hereinby reference.

The effective neuropeptides for use in the present invention may beisolated from the above-noted natural sources or may be readily preparedsynthetically, such as by solid phase peptide synthesis techniques. Forexample, the synthesis can be commenced from the carboxyl terminal endof the peptide by coupling the appropriate amino acid, e.g. L-arginine,L-isoleucine, L-phenylalanine or L-valine, to a suitable resin support,such as a p-methyl benzhydrylamine resin, a chloromethylated resin or ahydroxymethyl resin.

The coupling reaction may be carried out with the aid of a carboxylgroup activating compound, such as dicyclohexylcarbodiimide, and withthe α-amino group of the amino acid protected with a protecting group,such as t-butyloxycarbonyl (BOC), benzyl (BZL), p-methylbenzyl (MBZL),t-amyloxycarbonyl (AOC), tosyl (TOS), o-bromobenzyloxycarbonyl (BrZ),cyclohexyl (OHEX), or dichlorobenzyl (BZLCl₂). Following this couplingreaction, the α-amino protecting group is removed, such as by usingtrifluoroacetic acid in methylene chloride, trifluoroacetic alone or HClin dioxane, with the deprotection being carried out at a temperaturebetween about 0° C. and room temperature. Thereafter, each succeedingamino acid in the sequence is coupled in the same manner stepwise in thedesired order, culminating in the addition of the final amino acid(e.g., L-serine, L-asparagine, or L-glutamine) to obtain the desiredpeptide.

As an alternative to adding each amino acid separately to the reaction,some may be coupled prior to addition to the solid phase reactor. Eachprotected amino acid or amino acid sequence is introduced into the solidphase reactor in excess (about a three- or four-fold excess), and thecoupling may be carried in a medium of dimethylformamide:methylenechloride 1:1, or in dimethylformamide or methylene chloride alone. Thesuccess of the coupling reaction at each stage of the synthesis may bemonitored by the ninhydrin reaction.

After the final amino acid in the sequence has been coupled, thedeprotection step is carried out by treatment with a reagent such ashydrogen fluoride.

When a p-methyl benzhydryl amine resin has been used as the resinsupport, the peptide cleaved (by treatment with a reagent such ashydrogen fluoride) from the resin will be in the carboxyl terminal amideform. When a chloromethylated resin or a hydroxymethyl resin has beenused as the resin support, the peptide cleaved from the resin supportwill be in the form of the carboxyl terminal benzyl ester, which maythen be readily converted by methods well known in the art to providethe carboxyl terminal amide form of the peptide.

Therapeutically effective doses of CRF or its analogs in practicing thisinvention are at least about 0.01 μg/kg, more preferably from about 0.1to about 200 μg/kg, and most preferably are from about 0.1 to about 50μg/kg. A particularly preferred dose is about 1 to about 30 μg/kgadministered i.v. or subcutaneously. The dose may be infused slowlyintradermally or subcutaneously, or may be injected directly into anafflicted body part. When injected locally, doses of about 10 to about100 μg per local administration (i.e. about 0.1 to about 1 μg/kg bodyweight) are preferred.

The neuropeptides should be administered under the guidance of aphysician. Administration is preferably by intravenous, intradermal, orsubcutaneous injection. Administration can be within about two weeksbefore or after injury, preferably about two hours before deliberatelacerations of the musculature, brain surgery, or the like, andpreferably up to about three days after surgery or accidental injury.The drug is preferably delivered via the bloodstream, but localinjections into the cerebrospinal fluid, brain, or into the muscle canbe used for administration.

The active neuropeptide may be administered in combination with apharmaceutically acceptable carrier, such as isotonic saline, phosphatebuffer solution, or the like. Topical administration is not preferred,since CRF or an analog is a large molecule (e.g., 40 or 41 amino acids)and is not as efficiently delivered to the site of action as whenadministered by injection.

Although the peptides are generally water soluble as typicallysynthesized, they may be administered in the form of pharmaceuticallyacceptable non-toxic salts, such as acid addition salts. Illustrativeacid addition salts are hydrochloride, hydrobromide, sulfate, sulphate,acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate, orthe like.

An in vivo model of injury to study brain edema has been developed as areproducible edema model, which has the features of immediate corticaldamage followed by the subsequent development of brain edema. This modelis described by Chan et al., Brain Research, 277, pp. 329-337 (1983).The model uses rats which are anesthetized. A 2.5 cm incision is madeover the sagittal suture and the bone of the right hemisphere exposed. A60 mm² plate attached to a brass cup filled with dry ice-acetonemixture, with a temperature of -50° C., is applied to the rat skull forone minute. The animals are sacrificed at various intervals after theonset of cold-injury. A dye is administered intravenously before thefreezing. Cortical slices are then obtained of the brain.

EXAMPLE I

Sixteen male rats were randomly divided into eight pairs and one rat ineach pair received either saline or CRF (subcutaneously twice at 30μg/kg, 30 min and 10 min before cold injury). The animals wereanesthetized with sodium pentobarbital, 60 mg/kg intraperitoneally, andinjected with Monastral blue, 60 mg/kg intravenously. A cold probe wasapplied onto the skull for four minutes and the brains taken out onehour after cold injury. The staining of brain tissues with Monastralblue, a colloidal pigment that gets trapped between the albuminalsurface of the endothelial cell and the basement membrane, wasproportional to the degree of vascular leakage. The results from thefirst pair are shown as FIG. 1. Table I summarizes the data.

                  TABLE I                                                         ______________________________________                                        CRF and Freeze Injury to Brain                                                Treatment Area mm.sup.2                                                                             Intensity Lesion Size                                   ______________________________________                                        Saline    43.4 ± 1.7                                                                             2.2 ± 0.04                                                                           96 ± 4                                     CRF       20.8 ± 1.2                                                                             2.0 ± 0.05                                                                           43 ± 3                                     ______________________________________                                    

The size of the lesion, measured as area in mm², and the degree ofstaining intensity, were quantified using an image-analysis softwareprogram called JAVA (Jandel Corporation, San Rafael, Calif.). The stainintensity, given in arbitrary units, was internally calibrated usingMonastral blue solutions (1-30 mg/ml) placed on white filter paper.Values are mean ±S.E.M.

As can be seen visually from the shading of FIG. 1, the area andintensity of vascular permeability produced by cold injury was greatlyless for the brain of a rat treated in accordance with the invention byadministration of CRF than the brain of a rat treated with saline. Ascan be seen by the data of Table I, the lesion size of the CRF-treatedgroup was only 44% that of the saline-treated group.

An observer, unaware of the rat's pretreatment and asked to distinguishbetween more damaged brains from the less damaged brain, is able tocorrectly guess 8 out of 8 times the assignment of the brains to eitherthe saline or CRF group. Thus, the ability of CRF to suppress vascularleakage in the brain is shown.

EXAMPLE II

In a second set of experiments, the brain surface, namely the cerebralcortex, was injured by freezing and the water and sodium content weremeasured in the cerebral cortex and in the basal ganglia, a part of thebrain away from the freeze zone. The water and sodium content of thebrain tissue served as an index of brain edema. After freeze injury, thewater and sodium content of the cerebral cortex were evaluated relativeto non-frozen tissues. CRF administered in two separate doses of 30μg/kg subcutaneously, one dose 15 minutes before the injury and thesecond dose 90 minutes after the first dose, inhibited the two indicesof brain edema. Table II summarizes the data.

                  TABLE II                                                        ______________________________________                                                      WATER         SODIUM                                            TREATMENT     CONTENT %     mEq/kg dry                                        ______________________________________                                        CEREBRAL CORTEX                                                               Saline        87 ± 0.2   310 ± 9                                        CRF           77 ± 4*    271 ± 8*                                       BASAL GANGLIA                                                                 Saline        75 ± 1     213 ± 4                                        CRF           76 ± 1     215 ± 9                                        ______________________________________                                         Values are ±S.E.M., N = 5 animals per group                                *P < .05 vs Saline Controls                                                   Male SpragueDawley rats, 300-325 g, anesthetized with                         ketamineacepromazine, were injected saline or CRF (30 μg/kg s.c. 2x)       and injury to cerebral cortex was produced by applying a cold probe to th     skull for 1 min. Brain tissues were obtained 3 hr later and analyzed for      water and sodium content.                                                

As can be seen from the data of Table II, the saline treated rats hadincreased water content at the cerebral cortex injury and substantiallyincreased sodium with respect to the rats treated in accordance with theinvention with CRF. That is, since the water and sodium content of thebrain tissue served as an index of brain edema, the CRF treatedrats wereshown to have a suppressed vascular leakage due to cerebral cortexinjury; however, the inventive treatment did not affect the water orsodium content in the non-injured basal ganglia.

EXAMPLE III

Experiments were conducted on male Sprague-Dawley rats 243±15 gm (S. D.,Simonsen Labs., Gilroy, Calif.) anesthetized with sodium pentobarbital,60 mg/kg i.p. Monastral blue, 60 mg/kg i.v., was injected 0.2 ml/100 g,3 minutes before a 4 cm midline incision through the abdominal musclewall (celiotomy). Saline or CRF was administered to randomized pairswith N=8 rats per group. The size of the lesion, measured as area inmm², and its light intensity, were quantified using the JAVAimage-analysis software program. The light intensity, given in arbitraryunits, was internally calibrated using Monastral blue solutions (1-30mg/ml) placed on white filter paper. Values are mean±S.E.M. FIG. 2illustrates the respective amount of vascular permeability for one pairof rats (control and treated). Tables III and IV set out data from theseexperiments.

                  TABLE III                                                       ______________________________________                                        Dose-Response Data for CRF                                                    and Vascular Leakage After Celiotomy                                          Treatment  Area mm.sup.2                                                                             Intensity Lesions Size                                 ______________________________________                                        Saline     778 ± 34 2.1 ± 0.03                                                                           1624 ± 57                                 CRF 15 μg/kg                                                                          505 ± 16 2.0 ± 0.03                                                                           1031 ± 44                                 CRF 30 μg/kg                                                                          361 ± 18 1.9 ± 0.05                                                                            676 ± 47                                 CRF 60 μg/kg                                                                          257 ± 3  1.8 ± 0.05                                                                            468 ± 19                                 ______________________________________                                         CRF: inhected at various doses subcutaneously 30 minutes before a 4 cm        middle incision, tissues taken 0.5 hour after surgery.                   

                  TABLE IV                                                        ______________________________________                                        Long Duration of CRF Before Surgery                                           Treatment Area mm.sup.2                                                                             Intensity Lesion Size                                   ______________________________________                                        Saline    735 ± 32 2.1 ± 0.02                                                                           1547 ± 62                                  CRF       477 ± 31 1.9 ± 0.03                                                                            919 ± 51                                  ______________________________________                                         CRF: 30 μg/kg s.c. injected 2 hour before a 4 cm midline incision,         tissues taken 0.5 hour after surgery                                     

The celiotomy data illustrate the efficacious results from use of CRF inaccordance with the invention as a result of musculature injury. Thus,the data of Table III show that vascular leakage was reduced in a 5 dosedependent manner since the lesion size of the CRF-treated groups were63%, 42%, and 29% that of the saline-treated group, respectively.

Table IV shows that CRF can be administered even two hours beforemusculature injury and still significantly reduce vascular leakage,since the lesion size of the CRF-treated group illustrated by Table IVwas 59% that of the saline-treated group.

Increased vascular permeability occurs when blood vessels are exposed totoxic substances generated in injured tissues. These substances, calledinflammatory mediators, include chemicals such as serotonin, substanceP, bradykinin, neurotensin, inflammatory cytokines interleukin-1, tumornecrosis o factor (TNF), and histamine. It has previously been shownthat CRF will antagonize the edema-producing properties of thesemediators when injected into the rat pawskin. These mediators act onblood vessels principally in the skin and mucosa.

For example, in the class of inflammatory mediators are those calledplatelet-activating factors (PAF), which act not only on blood vesselsin the skin and mucosa, but also on small blood vessels in the lung andother visceral organs. PAF-acether is a prototype member of the PAFfamily. On a molar basis, PAF-acether is two to four orders of magnitudemore potent than any other currently known vasoactive substance. PAF arerapidly synthesized by inflammatory cells when responding to injury andincrease blood vessel permeability. PAF have been shown to be causallyrelated to a variety of adverse medical conditions and may account forthe pathologic and symptomatic processes of the disease state. Forexample, when bacteria are present in the bloodstream and producetoxins, the toxins stimulate the release of PAF and other factors, suchas interleukin-1 and tumor necrosis factor, which then cause toxicity toendothelial cells lining the blood vessels resulting in increasedvascular permeability throughout the various organs of the body, butespecially the lung, and produce various conditions, such as multipleorgan failure, pulmonary edema, and shock, which is manifested as a fallin blood pressure, blood volume, and hemoconcentration. Infection by thepresence of bacteria in the bloodstream (a condition known as sepsis) isbroadly-viewed as a trigger for the generalized inflammation of theendothelial cells lining the blood vessels and this condition can alsobe caused by non-septic inflammatory conditions such as pancreatitis,ischemia, multiple trauma, and tissue injury, hemorrhagic shock, andimmune-mediated organ injury. A group of researchers has recentlyestablished the designation "systemic inflammatory response syndrome"for use to describe these related conditions. (American College of ChestPhysicians--Society of Critical Care Medicine Consensus Conference.Definitions for sepsis and organ failure and guidelines for the use ofinnovative therapies in sepsis. Chest, 101, pp. 1644-1655 (1992). Thecriteria for the clinical diagnosis of systemic inflammatory responsesyndrome have been described by R. C. Bone (J. Amer. Med. Assoc., 268,pp. 3452-3455 (1992).)

Although one condition for which the invention is designed to treat isendotoxin shock, which is typically a gram-negative bacterial infectionand is manifested as a fall in blood pressure, blood volume, andhemoconcentration, patients with severe systemic inflammatory responsesyndrome should not be assumed to have a gram-negative bacterialinfection because a recent study suggests that less than half thepatients in a comparative analysis of patient characteristics in fourstudies had bacteremia. Bone, JAMA, 268, pp. 3452-3455 (Dec. 23/30,1992).

Another condition in which PAF or other inflammatory mediators have beenimplicated is in the deterioration of organs after they have beenremoved from the body. This deterioration is a natural consequence ofincreased water permeation of healthy tissues.

Thus, an agent capable of antagonizing inflammatory mediators, such asPAF, will have therapeutic benefit in treating the severe inflammatorycondition now sometimes designated as "systemic inflammatory responsesyndrome" and in the preservation of organs, such as for transplant ofkidneys, heart, liver, and lungs, and for amputated limbs or digitsprior to re-attachment to the body. In such uses the vasculature of theorgans to be transplanted are preferably perfused with a solutioncontaining about 5 to about 500 μg of CRF or CRF analogs.

The data of Table V shows that the increased vascular permeabilityproduced by PAF-acether is antagonized by CRF.

                  TABLE V                                                         ______________________________________                                        CRF Inhibits Vascular Leakage Produced by PAF-Acether                         Treatment Area mm.sup.2                                                                             Intensity Lesion Size                                   ______________________________________                                        Saline    327 ± 22 1.8 ± 0.04                                                                           589 ± 43                                   CRF       188 ± 16 1.4 ± 0.05                                                                           260 ± 26                                   ______________________________________                                         CRF: 30 μg/kg s.c. injected 30 minutes before subcutaneous injection o     PAFacether (1 μg/0.1 ml/rat) into the abdomen; muscle removed 0.5 hour     later.                                                                   

The muscle lesion size of the CRF-treated group was 44% that of thesaline-treated control group, illustrating the beneficial use of theinvention in conditions such as where patients are experiencingendotoxin shock due to PAF.

EXAMPLE IV

The neutropenic rat model of sepsis, designed to mimic the systemicinflammatory response syndrome in humans, is described by Collins et al.from The Journal of Infectious Diseases, 159:6 (June 1989) and TheJournal of Clinical Investigation, 88:885 (September 1991). The whiteblood cells of rats are artificially decreased and then the animals areinfected with the bacteria Pseudomonas. The animals die fromoverwhelming infection within 7 to 9 days of infection and examinationof the tissues with histological and pathological techniques reveal thecharacteristic organic manifestations of the systemic inflammatoryresponse syndrome, namely, organ edema and tissue necrosis.

In a typical experiment, 10 rats serving as controls received saline and15 rats received CRF at a dose of 100 μg/kg intravenously, given twice aday for four days after the onset of fever. At the end of 8 days, all ofthe animals in the control group were dead (100% mortality). Bycontrast, 60% (9/15) of the CRF group were still alive at day 11 whenthe experiment was concluded. The difference between the saline and CRFtreated groups were statistically significant (P<0.002). This data isillustrated by FIG. 3, where the vertical axis is percent survival andthe horizontal axis is days.

It is to be understood that while the invention has been described abovein conjunction with preferred specific embodiments, the description andexamples are intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims.

It is claimed:
 1. A method of preserving organs for transplantcomprising:perfusing the organ's vasculature with about 5 to about 500μg of corticotropin-releasing factor.